A.
SPECIFIC AIMS The proposed studies have two objectives: to further our understanding of the biology of posttranscriptional tRNA modifications and to explore the utility of the tRNA modifications as biomarkers of the pathophysiology of chronic inflammation leading to cancer and other diseases. There are dozens of different nucleobase structures in tRNA, yet the precise function of most of these modifications is unknown. We have recently demonstrated that enzyme-catalyzed methylation of a modified uridine (mcm5U) in tRNA plays a role in codon- specific translational control after DNA damage, and we have preliminary data demonstrating that the levels of mcm5U and other tRNA modifications specifically change after exposure to a variety of genotoxins. We hypothesize that the levels and patterns of enzyme-catalyzed tRNA modifications can be used as biomarkers of toxicant exposures and pathophysiologies. Inherent to our hypothesis is the novel proposal that enzyme-catalyzed tRNA modifications are dynamic components of damage signaling pathways. We will use biomarker studies to test our hypothesis, to establish enzyme-catalyzed tRNA modifications as components of damage signaling pathways, and to better understand the temporal order and protein systems that catalyze modifications into human tRNA. To accomplish our objectives, we will perform quantitative assessments of the spectrum of tRNA species, tRNA nucleobase 2? modifications, and the expression of tRNA-related genes, before and after exposure of cells to alkylating agents and reactive oxygen and nitrogen species (RONS). The combinatorial potential of more than 23 genes specific to human tRNA modification systems, 63 tRNA species distinguishable by array techniques, and more than 25 nucleobase modifications will be assessed to test our proposal that tRNA-centric indices are highly specific biomarkers with a large dynamic range. Further, tRNA-centric indices will be used to clarify the mechanistic basis by which modifications are incorporated into tRNA and to highlight the role of tRNA in the cellular response to noxious stimuli and pathophysiological conditions such as inflammation.
Aim 1 : Develop methods to quantify the spectrum of RNA nucleobase 2? modifications. The objective of this aim is to develop analytical methods for quantifying enzyme-catalyzed nucleobase modifications in tRNA. We will use two mass spectrometric approaches to quantify RNA modifications: global or untargeted surveys and targeted quantification of specific nucleosides. The strategy here is to use the global approach to define the spectrum of modifications undergoing quantitative changes during cell stress and to correlate modifications with specific enzymes and pathways. If an exposure """"""""signature"""""""" is identified in global studies, the individual modifications can be quantified with greater sensitivity by a targeted strategy. The methods will be developed and validated with yeast tRNA and then applied to tRNA from mouse and human cells.
Aim 2 : Assessment of tRNA-centric measures as agent-specific biomarker signatures of exposure in cells. The objectives of this cell-based aim are to apply the methods of Aim #1 to assess tRNA as a biomarker of cellular stress and to gain mechanistic insight into human tRNA modification systems. Further, these studies will provide a library of tRNA-specific changes that can be translated into in vivo studies. First, we will perform detailed dose-response and time course studies for changes in modified nucleosides after exposure of human HEK293 cells to the DNA damaging agents methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). The results will guide subsequent measurements of the levels of (1) transcripts corresponding to tRNA modification enzymes, (2) individual tRNA species, and (3) tRNA modifications for determination of tRNA- centric exposure signatures. To test our hypothesis and as a benchmark for the SJL mouse inflammation studies of Aim #3, we will quantify tRNA-centric indices in cultured SJL cell lines exposed to four chemical mediators of inflammation (H2O2, nitric oxide, peroxynitrite and HOCl). In all studies, the resulting quantitative data will be computationally mined to identify biomarker signatures specific to each agent. Further, we will use our biomarker data to highlight a role for tRNA modifications in damage signaling and to provide mechanistic insight into human tRNA modification systems.
Aim 3 : Use a mouse model of inflammation to demonstrate that tRNA modifications have in vivo biomarker potential. The methods applied to cells in Aim #2 and the resulting models will now be translated to the SJL mouse model of nitric oxide over-production and inflammation. The goal here is to quantify the transcripts corresponding to tRNA modification enzymes and to characterize the spectrum of tRNA modifications from tissues exposed to the reactive oxygen and nitrogen species arising from inflammation in the SJL mice. The resulting patterns will be compared to control animals to test our hypotheses that tRNA- centric measures can be used as highly sensitive in vivo biomarkers of chronic inflammation and that tRNA- centric measures can be used to assess the chemistry that occurs at sites of inflammation in vivo.
RNA Modifications as Biomarkers of Environmental Stress and Inflammation The proposed studies will be used to further our understanding of the biology of posttranscriptional RNA modifications and to explore the utility of RNA modifications as biomarkers of the pathophysiology of chronic inflammation. The long term potential of the proposed research is the use of modified nucleosides to predict risk and clinical outcomes related to exposures and inflammation induced cancers.
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